The major unresolved problem in biology is to understand the mechanisms by which transcription is regulated. Such understanding can be accomplished by establishing structural, thermodynamic and kinetic determinants of regulatory processes. This proposal is focused on structural aspects of regulation of transcription by transcription activators. The model system, which will be used in this work, is E coli RNA polymerase (RNAP). In our research we will use a combination of fluorescence spectroscopy, protein chemistry and molecular biology, and we will also take advantage of a novel experimental approach developed recently in our laboratory to map protein domains involved in macromolecular interactions. Our long term goals are to understand how RNAP, depending on the structure of the promoter and depending on the presence or absence of transcription activators readjusts the set of available interactions to perform its biological function. To achieve our goals we propose; aim #1 Studies on the molecular architecture of activator-RNAP-class I and class II promoter complexes to determine contact domains of RNAP involved in protein-protein and protein-DNA interactions, to determine the distances between functional units of the activator-RNAP-promoter complex and their modulation by promoter structure. aim #2 Studies on the role of a specific alpha subunit-DNA contact in RNAP activation by the activator protein to determine whether activator protein-alpha subunit and alpha-subunit-DNA interactions cooperate to produce active form of RNAP. aim #3 Studies on the communication between contact domains of RNAP to determine how DNA binding and promoter melting properties of RNAP are modulated. The results of this study and the novel methodology developed will have general applicability to studies of transcriptional regulation of prokaryotic and eukaryotic systems. Understanding regulation of transcription at the molecular level is of crucial importance for understanding development and the pathogenesis of abnormal development and neoplasia.